Proton pumps of the plasmalemma of the yeast Rhodotorula gracilis Their coupling to fluxes of potassium and other ions

Abstract

(1) Intact cells of the obligatory aerobic yeast Rhodotorula gracilis (glutinis) generate a difference of the electrochemical proton potential ( Δμ H + ) across the plasmalemma. In the range from pH 4.0 to 7.0 its value remains close to 12 kJ/mol. At pH 4.0 it is composed of the pH difference (inside alkaline) alone, at pH 7.0 of the membrane potential alone. (2) Both components of Δμ H + are generated by an active process, as shown by their rapid dissipation under anaerobic conditions. (3) In order to find out by which type of mechanism Δμ H + is generated the effect of a number of inhibitors of transport-ATPases (among them ouabain, triphenyltin chloride, quercetin, oligomycin, venturicidin, dicyclohexylcarbodiimide, Dio-9) were tested both on the generation of the membrane potential and on the extrusion of protons either in the absence or the presence of potassium ions. We found that all three processes were inhibited by Dio-9 and dicyclohexylcarbodiimide, which are specific for H +-ATPases. Triphenyltin chloride inhibited the K +/H +-exchange without having any effect either on the extrusion of H + alone or on the membrane potential. (4) Dicyclohexylcarbodiimide and Dio-9, but not triphenyltin chloride inhibited at pH 4.0 the active transport of sugars. This class of substrates has been shown earlier to be transported by an electrogenic H + symport driven by Δμ H + across the cell membrane. (5) Neither the rate of respiration nor the intracellular level of ATP were significantly decreased by any of these inhibitors (except for venturicidin). (6) We conclude that in Rhodotorula gracilis the difference of the electrochemical potential of H + is created by an electrogenic proton pump, presumably in ATPase. The extrusion of protons in exchange against potassium is catalyzed by a different energy-dependent but electroneutral system. This conclusion is based on the observation that the H +/K + exchange does not work under conditions where the membrane potential is large, and vice versa.